1. Field of the Invention
The present invention relates generally to the fields of immunology, viral diseases and cancer immunity. More particularly, it concerns the induction of immunity to viruses or cancers by the expression of nucleic acids encoding a granzyme inhibitor in cytotoxic T-lymphocytes (CTLs) or by the provision of a granzyme inhibitor to a CTL. Specifically, the invention provides methods for the induction of immunity, methods for the induction of long-term protective immunity, and methods for preventing and alleviating chronic viral infections and/or treating and preventing cancers by providing granzyme inhibitors to a patient.
2. Description of Related Art
Diseases caused as a result of viral infections pose a worldwide public health problem. For example, infection by the human immunodeficiency virus (HIV), alone is currently responsible for an estimated 36.1 million people worldwide living with acquired immune deficiency syndrome (AIDS). HIV is exemplified by several viral strains such as HIV-1 and HIV-2. In addition to HIV, other viruses such as, HCV, HTLV-1, HTLV-2, hepatitis G, enterovirus, dengue fever virus, rabies virus, etc., also result in a wide variety of pathologies.
Cancer is another worldwide health problem and is a leading cause of death. In the United States alone cancer strikes one in two men and one in three women. Cancer cells evade recognition by the immune system and continue to proliferate, eventually leading to metastasis and death. The art presently lacks effective methods to enhance the immune system to combat cancers.
Cytotoxic T-cells (CTLs) are the major protective mechanism against intracellular pathogens (especially viruses) and tumor/cancer cells, and exert their protection by killing infected cells. CTLs provide protection against pathogens by using cytolytic molecules such as perforin and granzyme B to kill infected cells. Perforin facilitates the entry of active granzyme B into the cytoplasm where it initiates a cascade of biochemical changes that result in apoptosis of a infected cell or a tumor cell.
The engagement of T-cell receptors (TCRs) on naive CD8 cells by antigen peptide-class I major histocompatibility complexes (pMHC) leads to the proliferation and differentiation of CD8 cells into CTLs (Zinkernagel and Doherty, 1974). Cytolysis is initiated by the formation of antigen-specific conjugates that result in the lysis of pMHC presenting target cells (Zinkernagel and Doherty, 1974; Kagi et al., 1996). In CTLs, inactive granzyme B and perforin are stored in cytoplasmic granules. During cytolysis, perforin facilitates the entry of active granzyme B into the cytoplasm of targets where it initiates a cascade of biochemical changes that result in apoptosis (Kagi et al., 1994; Huesel et al., 1994).
After this effector phase, a period of death ensues during which activated CTLs undergo activation induced cell death (AICD) (Razvi and Welsh, 1995). The third phase of the CD8 response is characterized by the appearance of memory cells that persist for many years and facilitate accelerated responses upon re-exposure to antigen such as viral antigens or tumor antigens. This is due to both an increase in the frequency of antigen-specific T-cells and to qualitative changes that allow them to respond to antigen more effectively than naive cells (antigen-hyperactivity) (Ahmed and Gray, 1996).
After a CTL has damaged its target cell it can recycle to lyse new targets, thus, during the lytic process CTLs are spared from autolysis (Berke et al., 1972; Berke and Amos, 1973). Consistent with this are observations that CTLs appear to be more resistant to cytolysis than experimental targets in vitro, suggesting a mechanism by which a CTL may protect itself from self-injury (Blakely et al., 1987; Kranz and Eisen, 1987; Zanovello et al., 1989). However, under some circumstances CTLs can be killed by other CTLs, a process referred to as fratricide (Walden and Eisen, 1990; Huang et al., 1999).
It has been shown previously that post-effector CD8+ cells are the precursors of memory cells (Opferman et al., 1999). This is consistent with the earlier finding that the number of CTLs that arise in response to viral infection (clonal burst size), determines the size of the pool of anti-viral memory CD8 cells (Gagliardini et al., 1994). However, the mechanism by which some CTLs escape AICD and give rise to memory cells remains unclear.
The present inventors have examined the role of cytolytic behavior on the cell fate decision made by functionally competent CTLs (Opferman et al., 2001). Memory cells were only generated from populations of CTLs that had killed little because CTLs underwent apoptosis during cytolysis through the action of effector molecules stored in lytic granules. This is consistent with observations that perforin plays a role in inducing the AICD of CTLs (Spaner et al., 1998; Spaner et al., 1999; Stepp et al., 1999; Matloubian et al, 1999). Thus, the perforin/granzyme pathway also plays a role in memory cell development.
A possible mechanism for ensuring CTL survival during cytolysis may involve the action of endogenous serine-protease inhibitors (serpins), (reviewed in Bird, 1999). More recently a human ova-serpin, PI9, has been identified in T-cells (Sun et al., 1996). P19 is a potent inhibitor of granzyme B and protects cells in vitro from perforin/granzyme killing but not Fas-mediated death (Bird et al., 1998). Although a homologue of P19, known as SPI6, has been identified in murine lymphocytes, its potential anti-apoptotic role remains to be determined (Sun et al., 1997).
Infections with certain viruses induce high zone tolerance when the intact immune system is confronted with an excessive viral burden (Ahmed et al., 1984; Moskophidis et al., 1993 a and b). High zone tolerance is thought to be due to excessive stimulation by antigen, which induces the clonal exhaustion of CTLs and attenuates the development of anti-viral memory. The end result is chronic viral persistence and in some cases generalized immunosuppression of T-cell responses (Wu-Hsieh et al., 1988). The molecular mechanism that gives rise to the attenuation of CTL responses in clonal exhaustion is unclear. Studies have implicated the CTL effector molecule perforin in the induction of CTL apoptosis that contributes to clonal exhaustion (Matloubian et al., 1999).
The effectiveness of the treatment of chronic infections by the adoptive transfer of CTLs has been hampered by ineffective generation of persistent memory CD8 cells (Brodie et al., 1999; Tan et al., 1999). In addition, the highly desirable induction of protective CTL-memory by vaccination has proved difficult (McMichael, 1998). Therefore, there is need for a better understanding as to how CTLs can eliminate virus but at the same time escape AICD. In addition, the art presently lacks an effective method to induce the immune system to provide long-term protection from viruses and other pathogens that require CTL-memory cells for elimination. Furthermore, with respect to cancers, the art lacks methods to induce CTL-mediated immunity against cancer cells.